s2fieldsofstudy: Biology Medicine

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Deregulated Expression of Circular RNAs Is Associated with Immune Evasion and Leukemia Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Circular RNAs (circRNAs) are a novel class of epigenetic regulators that participate in leukemogenesis. However, their roles in leukemia relapse after transplantation remain unclear. Methods: We defined the circRNAs profile of the bone-marrow-enriched CD34+ cells from ten acute myeloid leukemia (AML) patients after transplantation (five relapse [RE] and five continuous complete remission [CR]) and four healthy controls (HCs) by RNA-seq. Differentially expressed circRNAs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) in an independent cohort of six AML patients with pairwise samples at diagnosis and at relapse and six controls. Results: The bioinformatics analysis revealed a distinct circRNAs profile in relapse patients compared with controls (CR or HCs), while there was no significant difference between CR and HCs. Functional enrichment analysis demonstrated that mRNAs co-expressed with identified circRNAs were primarily involved in immune-related pathways, including the T cell receptor signaling pathway and lymphocyte differentiation. Moreover, we performed a protein–protein interaction network based on the immune-related genes and annotated 20 hub genes. The abnormal expression of hub genes was responsible for impairing T cell co-stimulation and activation, thus contributing to the immune escape of relapse blasts. We further constructed competing endogenous RNAs (ceRNA) regulatory networks based on immune-related genes and identified 10 key circRNAs that are associated with immune evasion. Six candidate circRNAs and their associated miRNA/mRNAs in the ceRNA network were randomly selected to be validated in another set by RT-qPCR. Conclusions: CircRNAs dysregulation may be involved in the immune evasion of relapse blasts and is associated with AML relapse. Our results identify several promising biomarkers and might provide novel insights into the biology of AML relapse post-transplantation.

Introduction
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment approach for hematological malignancies such as acute myeloid leukemia (AML) [1]. However, relapse remains a major challenge, and the clinical outcomes of relapsed patients have not significantly improved over the past decades [2]. The molecular mechanisms associated with post-transplantation relapse remain elusive, which has restricted more powerful therapeutic armamentarium against leukemia relapse.
In recent years, many researchers have studied the nature of AML relapse. There is growing evidence that the escape of control of allogenic immune responses may contribute to relapse after transplantation, including the downregulation of major histocompatibility complex (MHC) class II genes, the genomic loss of human leukocyte antigen (HLA) Genes 2022, 13,1986 2 of 16 and the enforcement of inhibitory checkpoints [3,4]. Moreover, previous studies have demonstrated that relapse after transplantation is in some cases associated with transcriptional changes, not the acquisition of previously unknown leukemia-specific mutations or structural variations, indicating potential epigenetic origins [5,6].
As an important epigenetic component, the discovery of noncoding RNAs (ncRNAs) has opened up broad prospects for cancer diagnosis, prognosis and treatment [7]. NcRNAs regulate gene expression by regulating transcription, post-transcriptional modification and translation, including microRNA (miRNA), long non-coding RNA (lncRNA) and circular RNA (circRNA) [8]. CircRNAs are a class of abundant, single-stranded ncRNA that result from the ligation of a downstream splice donor to an upstream splice acceptor which have been extensively studied in recent years [9,10]. In contrast with linear RNAs, circRNAs are characterized by exceptionally high stability, tissue-specific expression and evolutionary conservation. CircRNAs can regulate downstream gene expression by directly or indirectly altering transcription and translation through several mechanisms, including sponging miRNAs as competing endogenous RNAs (ceRNAs), serving as templates of translation, sequestering RNA-binding proteins (RBPs) and regulating RBPs interactions [11]. Accumulating evidence indicates that the dysregulation of circRNAs is implicated in a variety of physiologic and pathologic processes, including immune regulation and leukemogenesis [12][13][14]. Recent studies have supported that the dysregulation of immune-related pathways may be mainly responsible for the post-transplant relapse [4,15]. These findings prompted us to interrogate whether the expression of circRNA is altered during AML relapse after transplantation.
In the present study, we provide novel data on the global expression pattern of circRNAs during AML relapse after allo-HSCT. We identified key genes regulated by differentially expressed circRNAs compared with controls. Furthermore, we also constructed the ceRNAs regulatory network to evaluate the crucial circRNAs in buffering the regulatory activity of miRNAs and consequently their influence on the transcription of annotated protein genes. Our results show that the dysregulation of circRNAs may play an important role in the allogeneic immune responses and provide new insight into the underlying mechanisms of leukemia relapse after transplantation.

Patients and Samples
Given the important role of the post-transplantation immune environment in driving altered gene expression, we enrolled a discovery cohort (n = 14) and a validation cohort (n = 12) on the basis of sample availability. The discovery cohort consisted of 14 participants, including 5 AML patients with relapse after allo-HSCT (RE group), 5 patients with continuous complete remission for more than 2 years after transplantation (CR group) and 4 healthy controls (HC group). To further confirm the roles of key circRNAs identified in the discovery cohort in the AML relapse, we selected another validation cohort (n = 12) of 6 AML patients with pairwise samples at diagnosis and at relapse after allo-HCT and 6 controls (HCs = 3, CR = 3). All patients were diagnosed as AML-M5 according to the French-American-British (FAB) classification and underwent the first allo-HSCT between May 2019 and November 2020 in the Hematopoietic Stem Cell Transplantation Center of Blood Diseases Hospital, Chinese Academy of Medical Sciences. Relapse is morphologically diagnosed as more than 5% blast cells in the bone marrow (BM) or the appearance of extramedullary leukemic lesions. In the exploratory stage, we performed RNA-seq of BM samples from discovery cohort. Subsequently, RNA samples were isolated from an independent cohort of 12 participants (n = 6 per group) and were subjected to quantitative analysis for further validation. The clinical characteristics of these participants are shown in Table 1. The BM aspirations from all of the participants were harvested after informed consent approved by the Ethical Committee of Institute of Hematology & Blood Diseases Hospital. In detail, the BM samples were collected at the median of 160 days (interquartile range [IQR]: 127 to 243) and 124 days (IQR: 108 to 180) after transplantation from patients in the CR and RE group, processed to mononuclear cells with density centrifugation and cryopreserved in liquid nitrogen for further analyses. All relapse samples showed high expressions of the CD34 marker, and the AML blasts counts were approximately 80% in the clinical data. The HLA loss was negative in all of the relapse samples.

Isolation of CD34 + Cells and Flow Cytometry
The cryopreserved BM samples were thawed and the CD34 + cells were enriched by positive selection using CD34 microbeads and a magnetic cell-sorting system (Miltenyi). Briefly, the BM cell were suspended in 100 µL of PBS/0.5% BSA/2 mM EDTA solution and were filtered through a 30 µm nylon mesh to remove cell clumps. Human FcR blocking reagent and anti-CD34 microbeads were added and then incubated at 4 • C for 30 min. Subsequently, cells were washed and resuspended in 500 µL PBS solution and were passed through the MS separation column kept in the magnetic field to hold the CD34 + cells in the column. Then, the purity of the isolated CD34 + cells was evaluated by flow cytometry; the post-sort purity was routinely >95% ( Figure S1). In detail, 1 × 10 5 cells were suspended in 100 µL PBS and were stained with FITC-conjugated anti-CD34 (Biolegend) in the dark for 30 min on ice, and DAPI (1 µg/mL) was added to gated-out dead cells before running the samples. The samples were stained and analyzed on a FACS Canto II flow cytometer, and data was processed with FlowJo version 9 or 10 (Tree Star).

Whole Transcriptome Sequencing (WTS)
The total RNA from purified the CD34 + cells (2-3 × 10 6 ) was extracted using TRIzol reagent (Thermo Fisher) according to the manufacturer's protocol. The quality and quantity of total RNA was measured using Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). The RNA libraries were prepared with the TruSeq Stranded Total RNA Ribo-Zero Gold kit. An Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to test the RNA integrity, and samples with an RNA integrity number (RIN) ≥7 were subjected to subsequent analysis. Small RNA (miRNA) and RNA (circRNA, lncRNA and mRNA) library construction was conducted and sequenced on the Illumina Hiseq2500 platform at an average sequencing depth of 140 million reads per sample. CircRNAs were detected and quantified by CirComPara v0.6 using 9 backsplice detection methods and default parameters.

Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR)
In the validation cohort, 6 AML patients with pairwise samples at diagnosis and at relapse after allo-HCT and 6 controls (HCs = 3, CR = 3) were enrolled for quantitative analysis. Briefly, total RNA was extracted from primary BM CD34 + cells with TRIzol reagent and then first-strand cDNA was synthesized with a PrimeScript RT reagent Kit with gDNA Eraser (TAKARA, Otsu, Japan) according to the manufacturer's instructions. RT-qPCR for the miRNAs was performed on cDNA generated from 0.5 µg total RNA according to the manufacturer's protocol (Ribobio). U6 was used for miRNAs template normalization and GAPDH was used for circRNAs and coding genes template normalization, respectively. Quantitative validation was performed using a Fast SYBR Green Master Mix (Roche, Basel, Switzerland) on a QuantStudio 5 real-time PCR detector (ThermoFisher, Waltham, MA, USA) for DE circRNAs/miRNAs/mRNAs identified in the ceRNAs network. All primers used in this study are listed in Supplemental Table S1.

Identification of Differentially Expressed Genes
Purified CD34 + blasts or hematopoietic stem progenitor cells (HSPCs) from all patients and HCs were analyzed after quality control analysis. RNA sequencing reads were mapped to the GRCh38.p7 obtained from NCBI. Gene expression was normalized to fragments per kilobase million (FPKM) and analyzed further by log 2 (FPKM+1) transformation. Differential expression genes were determined by DEseq2 and |log 2 Fold change (FC)| > 1 with a p value < 0.05 as the threshold for statistical significance. The absolute value of a Pearson correlation coefficient >0.99 was defined as co-expression between differential expressions of circRNAs and mRNAs (DE circRNAs and DE mRNAs) according to the expression levels.

Functional Annotation and Enrichment Analysis
Gene ontology was conducted to annotate the co-expressed mRNAs with terms under the biological process (BP) (http://www.geneontology.org, accessed on 22 April 2022). The analyses of the signaling pathways with enriched mRNAs were based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg, accessed on 1 May 2022).

CeRNA Regulatory Network Analysis
The target miRNAs of DE circRNAs and co-expressed mRNAs were predicted by the CircNet, TargetScan and miRTarBase databases. Any overlapping of the same miRNAs was considered to be potential circRNA-miRNA-mRNA interactions. The ceRNA regulatory network was visualized using the Cytoscape software.

Statistics
The results are summarized as median ± interquartile range (IQR) where appropriate. The p values are two-sided and a value < 0.05 is considered statistically significant. All analyses were performed with Graph Pad Prism version 8.0 (Graph Pad, San Diego, CA, USA).

Overview of Global circRNAs Profiles in AML Patients with Relapse after Transplantation
To explore the potential effect of circRNAs on post-transplant relapse, we first performed RNA-seq on purified BM CD34 + cells from AML patients who received first allo-HSCT (CR and RE, N = five per group) and healthy controls (HC, N = four) in the discovery set ( Figure 1). In general, we identified on average 9880 different circRNAs per sample in the RE group, 9562 circRNAs in the CR group and 9500 circRNAs in the HC group. The principal component analysis indicated distinct circRNAs profiles among the groups ( Figure S2). the RE group, 9562 circRNAs in the CR group and 9500 circRNAs in the HC group. The principal component analysis indicated distinct circRNAs profiles among the groups (Figure S2).

CircRNAs Expression Specifically Correlates with Post-Transplant Relapse
To elucidate the unique expression profile of circRNAs from relapse blasts, we conducted statistical analysis following the criteria as mentioned above (|log2Fold change (FC)| > 1 and p value < 0.05). The circRNAs expression profile in the RE group significantly differed from those in the CR and HC groups ( Figure 2A). In contrast, there was not a clear distinction in circRNAs expression between patients in the CR and HC groups (Figure S3). A total of 5314 DE circRNAs (3057 upregulated and 2257 downregulated) were identified between the RE and CR groups ( Figure 2B,C). For relapse patients, there were 1292 DE circRNAs (674 upregulated and 618 downregulated) versus controls ( Figure  2D,E). In summary, these data demonstrate that striking differences in the expression of circRNAs expression exist in relapse blasts, supporting the important role of circRNAs in post-transplant relapse.
Among these DE circRNAs, 5665 circRNAs (99.3%) have been identified in circBase [16], and the others (0.7%) are novel ( Figure 3A). The genome distribution of DE circRNAs reveals that the majority of the identified candidates (5226/5665, 92.3%) are transcribed from protein-coding exons ( Figure 3B). As is consistent with a previous study [17], the DE circRNAs were widely distributed among chromosomes, and most of the identified circR-NAs were located on the chromosomes 1 and 2 ( Figure 3C). Moreover, the lengths of most exonic circRNAs were around 200-450 nucleotides ( Figure 3D).

CircRNAs Expression Specifically Correlates with Post-Transplant Relapse
To elucidate the unique expression profile of circRNAs from relapse blasts, we conducted statistical analysis following the criteria as mentioned above (|log 2 Fold change (FC)| > 1 and p value < 0.05). The circRNAs expression profile in the RE group significantly differed from those in the CR and HC groups ( Figure 2A). In contrast, there was not a clear distinction in circRNAs expression between patients in the CR and HC groups ( Figure S3). A total of 5314 DE circRNAs (3057 upregulated and 2257 downregulated) were identified between the RE and CR groups ( Figure 2B,C). For relapse patients, there were 1292 DE circR-NAs (674 upregulated and 618 downregulated) versus controls ( Figure 2D,E). In summary, these data demonstrate that striking differences in the expression of circRNAs expression exist in relapse blasts, supporting the important role of circRNAs in post-transplant relapse.
Among these DE circRNAs, 5665 circRNAs (99.3%) have been identified in circBase [16], and the others (0.7%) are novel ( Figure 3A). The genome distribution of DE circRNAs reveals that the majority of the identified candidates (5226/5665, 92.3%) are transcribed from protein-coding exons ( Figure 3B). As is consistent with a previous study [17], the DE circRNAs were widely distributed among chromosomes, and most of the identified circRNAs were located on the chromosomes 1 and 2 ( Figure 3C). Moreover, the lengths of most exonic circRNAs were around 200-450 nucleotides ( Figure 3D).

Co-Expression Analysis of DE circRNAs and mRNAs and Functional Enrichment
Alterations in circRNA expression can result in an aberrant expression of genes that contribute to myeloid leukemogenesis [8,18]. Thus, we constructed the circRNA-mRNA co-expressed network based on the correlation analysis (|PCC| > 0.99) between DE circR-NAs and DE mRNAs in the relapse samples versus the HCs. The co-expression analysis revealed that 311 DE mRNAs (98 upregulated and 213 downregulated) had a strong correlation with 153 DE circRNAs (90 upregulated and 63 downregulated) between the RE and CR groups ( Figures 4A and S4). In the RE group, 166 DE mRNAs were co-expressed with 239 DE circRNAs compared with the HCs (Figures 4B and S5).

Co-Expression Analysis of DE circRNAs and mRNAs and Functional Enrichment
Alterations in circRNA expression can result in an aberrant expression of genes that contribute to myeloid leukemogenesis [8,18]. Thus, we constructed the circRNA-mRNA coexpressed network based on the correlation analysis (|PCC| > 0.99) between DE circRNAs and DE mRNAs in the relapse samples versus the HCs. The co-expression analysis revealed that 311 DE mRNAs (98 upregulated and 213 downregulated) had a strong correlation with 153 DE circRNAs (90 upregulated and 63 downregulated) between the RE and CR groups ( Figures 4A and S4). In the RE group, 166 DE mRNAs were co-expressed with 239 DE circRNAs compared with the HCs (Figures 4B and S5).
To clarify the potential functions of identified DE circRNAs, we conducted GO and KEGG pathway analyses based on the co-expressed genes. Of note, the co-expressed genes in both pairs were enriched in similar pathways. The KEGG analysis revealed the top 10 enriched pathways among these DE mRNAs, including cytokine-cytokine receptor interaction, T cell receptor-signaling pathway and T cell differentiation ( Figure 5A,B). In the GO analysis, DE mRNAs related to biological processes that were mainly enriched in the immune response, immune system process, lymphocyte differentiation and leukocyte migration were identified ( Figure 5C,D).  To clarify the potential functions of identified DE circRNAs, we conducted GO and KEGG pathway analyses based on the co-expressed genes. Of note, the co-expressed genes in both pairs were enriched in similar pathways. The KEGG analysis revealed the top 10 enriched pathways among these DE mRNAs, including cytokine-cytokine receptor interaction, T cell receptor-signaling pathway and T cell differentiation ( Figure 5A,B). In the GO analysis, DE mRNAs related to biological processes that were mainly enriched in the immune response, immune system process, lymphocyte differentiation and leukocyte migration were identified ( Figure 5C,D).

CircRNAs Dysregulation Correlates with Immune Evasion during Post-Transplant Relapse
Several previous studies have documented that immune evasion plays a crucial role in mediating relapse after transplantation [15,19]. Consistently, GO analysis has suggested that co-expressed DE mRNAs are primarily involved in immune-related pathways in our study, such as immune response and immune system process. Notably, our results demonstrate downregulation in T cell co-stimulation molecules (HLA class II regulators [CIITA, IRF8, IRF4], the T cell receptor-signaling pathway [CD247, CD3E, CD3G, ITK], activated molecules [LAT2, TNFSF4, IFNG, IL15]) and the up-regulation of inhibitory T cell ligand (PVR) in the RE group, which are known to drive relapsed AML cells to evade from immune control ( Figure 6A,B). Our results indicate that DE circRNAs are implicated in promoting the immune-evasion of leukemia cells by regulating the expression of immune-related genes, and thus contribute to post-transplantation relapse. In our subsequent analysis, we focused more attention on these identified DE circRNAs and mRNAs.
To further explore the interaction between immune-related genes, we constructed protein-protein interaction (PPI) networks based on the identified DE mRNAs, which were involved in the immune response, immune system process, lymphocyte differentiation and leukocyte migration ( Figure 6C). A total of 20 DE mRNAs predicted by cyto-Hubba algorithms were identified as hub genes, such as CD247, PVR, IRF8, CD79B, CD70 and TNFSF4 (Table 2).

CircRNAs Dysregulation Correlates with Immune Evasion during Post-Transplant Relapse
Several previous studies have documented that immune evasion plays a crucial role in mediating relapse after transplantation [15,19]. Consistently, GO analysis has suggested that co-expressed DE mRNAs are primarily involved in immune-related pathways in our study, such as immune response and immune system process. Notably, our results demonstrate downregulation in T cell co-stimulation molecules (HLA class II regulators [CIITA, IRF8, IRF4], the T cell receptor-signaling pathway [CD247, CD3E, CD3G, ITK], activated molecules [LAT2, TNFSF4, IFNG, IL15]) and the up-regulation of inhibitory T cell ligand (PVR) in the RE group, which are known to drive relapsed AML cells to evade from immune control ( Figure 6A,B). Our results indicate that DE circRNAs are implicated in promoting the immune-evasion of leukemia cells by regulating the expression of immunerelated genes, and thus contribute to post-transplantation relapse. In our subsequent analysis, we focused more attention on these identified DE circRNAs and mRNAs.
To further explore the interaction between immune-related genes, we constructed protein-protein interaction (PPI) networks based on the identified DE mRNAs, which were involved in the immune response, immune system process, lymphocyte differentiation and leukocyte migration ( Figure 6C). A total of 20 DE mRNAs predicted by cytoHubba algorithms were identified as hub genes, such as CD247, PVR, IRF8, CD79B, CD70 and TNFSF4 (Table 2).

Construction of the circRNA-miRNA-Immune-Related mRNA Regulatory Network
Accumulating evidence indicates that circRNAs can indirectly regulate mRNA expression by sponging miRNA to relieve the repression of their target genes [20,21] To elucidate the role of circRNA-miRNA-mRNA interactions (ceRNA) during leukemia relapse, we constructed a ceRNA regulatory network depending on immune-related genes and their coexpressed circRNAs. We identified 15 circRNA-miRNA pairs and 27 miRNA-mRNA pairs using the CircNet, TargetScan and miRTarBase databases. The ceRNA network (including 10 circRNAs, 14 miRNAs and 16 mRNAs) was built by integrating with these relationship pairs ( Figure 6D). As shown in the ceRNA network, 10 key circRNAs might act as potential ceRNAs that sponged miRNAs to regulate the expression of immune-related genes. In sum, these data indicate that dysregulated ceRNA might be involved in the process of AML relapse after transplantation.

Validation of DE circRNAs by RT-qPCR
To verify the reliability of the DE circRNAs detected by the transcriptome sequencing, six DE circRNAs (three upregulated and three downregulated) and their associated miRNAs/mRNAs were randomly selected from our ceRNA regulatory network and further verified using RT-qPCR in the validation cohort. We selected six AML with pairwise samples purified at diagnosis and at relapse and six controls. Our results demonstrate that most of the DE circRNAs (5/6, 83.3%) were differentially expressed between pairwise diagnosis and relapse blasts ( Figure 7A,B). The results of RT-qPCR in the validation cohort are consistent with those of RNA-seq in the discovery cohort, suggesting that the alteration of DE circRNAs expression provides a clear distinction between AML relapse and controls.

Discussion
Relapse still represents the major cause of treatment failure, and up to 50% of AML patients finally relapse after allo-HSCT [2,22]. The dismal success of existing therapies underscores the importance of determining the underlying mechanisms of relapse and exploring novel treatment protocols. In the present report, we disclose the expression profile of circRNAs in BM CD34 + cells from AML patients with post-transplant relapse and controls, first providing the crucial circRNAs that are implicated in post-transplant relapse. Specifically, we revealed a distinct pattern of circRNA expression in relapse blasts compared with healthy or HSPCs in remission using RNA-seq. We subsequently identified the co-expressed mRNAs to investigate the potential regulatory roles of DE circRNAs. The CeRNA regulatory network analysis showed that hub genes regulated by DE circRNAs are primarily enriched in immune-related pathways, thus indicating their potential roles in immune evasion during relapse after transplantation. These results were validated and confirmed by RT-qPCR in an independent set for randomly selected circRNAs.
As emerging epigenetic molecules, circRNAs are widely defined as a subset of endogenous RNAs that regulate target genes by binding to miRNAs [23]. A single circRNA might have several binding sites for miRNAs and therefore bind to different miRNAs simultaneously, thus relieving the depression of their target genes [24]. As the "molecular sponge", circRNAs play important roles in several important physiological and pathological processes, including lymphocyte differentiation, immune responses, inflammation and oncogenesis [25][26][27]. In addition, circRNAs can contribute to the posttranscriptional regulation of other pathways, including absorbing RBPs to regulate the translation or production of proteins [28].
Immune surveillance plays a critical role in preventing leukemia's development and progression. Our study investigated the crosstalk between epigenetics and immune surveillance, providing the molecular insight into immune evasion during AML relapse. We have identified the unique expression profile of circRNAs in the relapsed AML cells. In contrast with control HSPCs (HC and CR group), we confirmed that mRNAs regulated by DE circRNAs in relapse blasts are primarily involved in immune-related pathways. As is consistent with previous reports [4,29], our results demonstrate that T cell co-stimulation is impaired by relapse blasts, which might abolish AML recognition from donor-derived T cells and outgrow into clinically evident relapse. Furthermore, recent analyses have elucidated that B cell's differentiation and function in the tumor microenvironment are typically associated with clinical outcomes and responses to immunotherapy [30]. Similarly, our results demonstrate altered expression profiles of the genes associated with B cell differentiation and the B cell receptor-signaling pathways (CD79B, BLNK, CD72, LYN, BTLA and IRF1) in the relapsed AML cells, indicating that B cells-mediated immune processes might have an effect on post-transplant relapse.
CircRNAs are demonstrated to function as miRNA sponges that regulate the downstream target genes. Several circRNAs have been linked to anti-tumor immune regulation through interactions with miRNAs [26,27,31]. Zhang et al. [32] demonstrated that hsa_circ_0020397 could upregulate the expression of PD-L1 by binding to miR-138 in colorectal cells, thus contributing to a tumor's escape from immune responses. Other studies have also identified that circRNAs are connected to immune checkpoints and immunotherapy in many solid tumors [33,34]. However, the functions of circRNAs in the transplant immunity during leukemia are less known. In this study, a total of 10 circRNAs, 14 miRNAs and 16 hub mRNAs were identified in the ceRNA network, which they might target in patients with AML relapse, a process known to be heavily involved in the immune-evasion of leukemia cells. Two of these ten circRNAs have been reported to be associated with the pathogenesis and development of tumors. Hsa_circ_0012152 (circRNF220), which has been previously reported to have elevated expression in pediatric primary AML [17], was upregulated in the relapse blasts and works as a sponge of miR-92a-1-5p to upregulate the expression of PVR in our study. Another circRNA, hsa_circ_0002768 (circMYLK), was significantly upregulated and contributed to malignant development via targeting miR-34a to enhance the CCND3 expression in bladder cancer cells [35]. However, circMYLK was downregulated in the relapse blasts and inhibited the expression of HLA class II regulators (CIITA). These contrasting results demonstrate the various roles of the same circRNA in the different diseases may be associated with variations in downstream target miR-NAs/mRNAs. Based on our bioinformatics analysis and RT-qPCR validation experiments, we first demonstrated that dysregulated circRNAs in the relapse samples modulate the expression of immune-related genes and are associated with allogeneic immune responses after transplant.
Notably, within downstream protein-coding genes controlled by DE circRNAs identified in the ceRNA network, we revealed some important transcripts that correlate with immune evasion. PVR (CD155), which is a novel immune checkpoint that binds to the T cell immunoreceptor with Ig and ITIM domains (TIGIT) to suppress immune responses, was highly expressed in the relapse blasts [4]. TNFSF4 (OX40L) is known as the cognate ligand for the tumor necrosis factor receptor OX40, which functions as a T cell co-stimulatory molecule [36]. Thus, the downregulation of TNFSF4 expression controlled by DE circRNAs has the potential to alleviate allogeneic T cell activity. In sum, deregulated circRNAs may exert their effects on immune escape through regulating immune-related genes, therefore contributing to leukemia relapse after transplantation.

Conclusions
In summary, this is the first study to investigate the comprehensive expression profile of circRNAs during AML relapse after allo-HSCT. Our data provides new insights into the molecular mechanisms associated with immune evasion during leukemia relapse after allo-HSCT, and therefore circRNAs may represent potential biomarkers for post-transplant relapse.

Limitations
This study has some limitations. First, the retrospective nature of the study, along with the small sizes of the BM samples, especially for diagnosis samples, have limited a more powerful conclusion. Furthermore, as our results were mainly obtained from bioinformatics models, further experimental studies are crucial to explore the exact role of these circRNAs in AML relapse. Despite the lack of the transcriptome information of diagnosis blasts, our findings provide clues for the roles of circRNAs in the AML development and relapse during transplantation. A larger scale analysis is warranted for more conclusive results.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes13111986/s1, Table S1. The primer sequences included in this study. Figure S1. The high viability and purity of CD34 + cells from the BM samples in the AML patients and healthy controls. Figure S2. Principal component analysis of circRNA expression profiles in AML patients with relapse (RE), remission (CR) and healthy controls (HC). Figure S3. CircRNAs expression profile in AML patients with remission (CR) and healthy controls (HC). Figure S4. DE circRNAs and mRNAs co-expression networks present in the CD34+ cells from patients with AML relapse versus remission. Figure S5. DE circRNAs and mRNAs co-expression networks present in the CD34+ cells from patients with AML relapse versus healthy controls. Institutional Review Board Statement: This study was approved by the institutional Medical Ethics Committee (KT2020002-EC-2).

Informed Consent Statement:
All patients or their legal representative provided written informed consent before participation.

Data Availability Statement:
The raw data supporting the conclusion of the present article will be made available by the author. Further inquiries can be directed to the corresponding author.


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Human Monocyte-Derived Dendritic Cells Are the Pharmacological Target of the Immunosuppressant Flavonoid Silibinin

Silibinin, a natural polyphenolic flavonoid, is known to possess anti-inflammatory, anticancer, antioxidant, and immunomodulatory properties. However, the effects of Silibinin on the maturation and immunostimulatory functions of human dendritic cells (DC) remain to be elucidated. In this study, we have attempted to ascertain whether Silibinin influences the maturation, cytokine production, and antigen-presenting capacity of human monocyte-derived DC. We show that Silibinin significantly suppresses the upregulation of costimulatory and MHC molecules in LPS-stimulated mature DC and inhibits lipopolysaccharide (LPS)-induced interleukin (IL)-12, IL-23, and TNF-α production. Furthermore, Silibinin impairs the proliferation response of the allogenic memory CD4 T lymphocytes elicited by LPS-matured DC and their Th1/Th17 profile. These findings demonstrate that Silibinin displays immunosuppressive activity by inhibiting the maturation and activation of human DC and support its potential application of adjuvant therapy in the treatment of autoimmune diseases.

Introduction
Silibinin, a natural polyphenolic flavonoid, is the major biologically active component of Silymarin isolated from the plant milk thistle Silybum marianum. Silibinin has been shown to have multiple protective biological activities due to its ability to modulate various cellular signaling pathways and to possess strong antioxidant, hepatoprotective, neuroprotective, anticancer, and antiviral activities [1]. It has been traditionally used for the treatment of various liver disorders [2] as well as of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and depression, due to its ability to increase the intracellular antioxidant defense and restore the redox status by changing the level of antioxidant enzymes [3]. The induction of apoptosis and its other abilities including cell growth inhibition, cell cycle arrest, an antiproliferative effect, and regulation of aberrant miRNA expression have supported the use of Silibinin as an anticancer and chemo preventive agent in a variety of cancers [4].
The biological actions of Silibinin include a broad spectrum of anti-inflammatory and immunosuppressive effects in a dose and time-dependent manner [5]. The potent anti-inflammatory effects of Silibinin have been shown to be related to the inhibition of several transcriptional factors such as the Nuclear Factor kappa B (NF-κB) and the Signal Transducer and Activator of Transcription 3 (STAT-3) which regulate several gene products involved in the immune response, inflammation, and cytokine production [6,7]. Silibinin is known to inhibit CD4 + T-cell activation and proliferation by inhibiting the Mitogen-activated protein (MAP) kinase pathway through T-cell receptor engagement [8] and to suppress the production of Th1-related cytokines (IL-2, IFN-γ, and TNF-α) by activated-PBMC from HCV-infected and uninfected subjects [9].
In agreement with these studies, we previously showed that Silibinin upregulates ERβ expression, induces apoptosis, inhibits proliferation, and reduces expression of the pro-inflammatory cytokines through ERβ binding in T lymphocytes from female and male healthy donors and patients with active Rheumatoid Arthritis [10]. Moreover, Silibinin has been also shown immunosuppressive effects in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis via inhibition of dendritic cell (DC) activation, and Th17 cell differentiation [11]. Silibinin has also been demonstrated to inhibit phenotypic and functional maturation of murine bone marrow-derived DC and Th1 polarization. In particular, it has been shown that Silibinin suppresses the expression of MHC class I and II and costimulatory molecules (CD80, CD86) and IL-12 production by murine bone marrow-derived DC [12].
DC play a key role in both the pathogenesis and treatment of several diseases. Therefore, DC are attractive targets for therapeutic manipulations of the immune response. However, the effect of Silibinin on human DC has never been investigated. Here, we show that Silibinin affects the LPS-induced maturation of human monocyte-derived DC by inhibiting the upregulation of costimulatory and MHC molecules and inflammatory cytokine production. Moreover, Silibinin treated DC fail to present alloantigens to memory CD4 + T lymphocytes which barely proliferate and the percentage of IFN-γ + and IL-17 + CD4 + T-cells is significantly lower.
Given the critical role of DC in the initiation and regulation of immune responses, these findings provide new insight into the immunosuppressive activity of Silibinin and support its potential application in the treatment of autoimmune diseases.

Silibinin Inhibits the Phenotypical Maturation of DC Induced by LPS Stimulation
DC play a pivotal role in the control of the innate and adaptive immune response. They reside in peripheral tissues and undergo a maturation process in response to diverse stimuli. Mature DC express high levels of major histocompatibility complex (MHC) molecules and co-stimulatory molecules that are important for antigen presentation to T lymphocytes.
To investigate the effect of Silibinin on DC, phenotype cells were cultured for five days in IL-4 and GM-CSF-enriched medium and stimulated overnight with LPS in the presence or absence of Silibinin at different concentrations (50 or 100 µM). Figure 1A shows that Silibinin added on the fifth day of culture did not alter the DC phenotype at either concentration. When added together with LPS at 50 µM, it inhibited only the upregulation of CD86 costimulatory molecule, whereas at 100 µM, it significantly suppressed the upregulation of co-stimulatory molecules CD80 and CD86 and of MHC class I and II induced by LPS stimulation. Furthermore, Silibinin decreased the novo-expression of CD83, a maturation marker for DC. These data indicate that Silibinin alters the DC maturation program which is needed for acquiring a fully professional antigen-presenting cell profile.

Silibinin Inhibits Proinflammatory Cytokine Production by LPS-Stimulated DC
The maturation process of DC induced by several stimuli leads to the production of a cascade of pro-inflammatory cytokines, which in turn control the induction and skewing of T-cell responses. LPS-stimulated DC produce huge amounts of IL-12 and IL-23 which are involved in Th1 and Th17 polarization, respectively, and of TNF-α cytokine which has pleiotropic effects on various cell types and is a major regulator of inflammatory responses. To investigate the effects of Silibinin on the pro-inflammatory cytokine profile of mature DC, we measured IL-12, TNF-α, and IL-23 secretion by DC stimulated with LPS in the presence of the flavonoid. Figure 1B shows a significant decrease in all three cytokines induced by a Silibinin treatment at 100 µM, thereby indicating an anti-inflammatory effect of Silibinin.

Mature DC Treated with Silibinin Do Not Present Alloantigens to Memory CD4 + T Lymphocytes
To investigate the effects of Silibinin on the antigen-presenting capacity of DC, we measured CD4 + T-cell response to alloantigens in a mixed leukocyte reaction. As shown in Figure 2, LPS-matured DC, as expected, induced a strong proliferation of allogeneic CD4 + T-cells and an increase in the percentage of IFN-γ and IL-17 producing CD4 + T-cells as compared to immature or Silibinin-treated DC. On the contrary, the same population of T-cells barely proliferated and the percentage of IFN-γ and IL-17 CD4 + T-cells was significantly lower in response to LPS-matured DC treated with Silibinin. These results demonstrate the immunosuppressive action of Silibinin on the most powerful antigen-presenting cells which play a crucial role in shaping the immune response in several diseases.

Mature DC Treated with Silibinin Do Not Present Alloantigens to Memory CD4 + T Lymphocytes
To investigate the effects of Silibinin on the antigen-presenting capacity of DC, we measured CD4 + T-cell response to alloantigens in a mixed leukocyte reaction. As shown in Figure 2, LPS-matured DC, as expected, induced a strong proliferation of allogeneic CD4 + T-cells and an increase in the percentage of IFN-γ and IL-17 producing CD4 + T-cells as compared to immature or Silibinin-treated DC. On the contrary, the same population of T-cells barely proliferated and the percentage of IFN-γ and IL-17 CD4 + T-cells was significantly lower in response to LPS-matured DC treated with Silibinin. These results demonstrate the immunosuppressive action of Silibinin on the most powerful antigen-presenting cells which play a crucial role in shaping the immune response in several diseases.

Monocyte Isolation and DC Generation
Peripheral blood mononuclear cells (PBMC) were purified from heparinized blood obtained by healthy donors on a density gradient (Ficoll-Hypaque, Lympholyte-H; Cedarlane, Paletta Court, ON, Canada).
Silibinin (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide, diluted in the culture medium, and used at 50 or 100 µM.

Flow Cytometric Analysis
Immature DC or LPS-stimulated DC in the presence or absence of Silibinin at 50 or 100 µM were plated into 96-well plates and washed once in phosphate-buffered saline (PBS) containing 1% FBS. The cells were incubated with Mabs at 4 • C for 30 min. Cells were then analyzed on a Beckman Coulter Gallios™ flow cytometer and data were analyzed with Kaluza Analysis Software v2.1 (Beckman Coulter, Brea, CA, USA).

Cytokine Production Analysis
After 5 days, the supernatant of DC cultures was removed, and the cells were washed and cultured in the presence or absence of 100 ng/mL LPS or Silibinin 100 µM for an additional 24 h. After 5 days, the supernatants from immature DC or LPS-stimulated DC cultures in the presence or absence of 100 µM Silibinin were harvested, filtered (0.2-µm filters), and stored at −80 • C. IL-23, IL-12p70, and TNF-α were measured by the specific enzyme-linked immunosorbent assay (ELISA) using the commercially available kit (R&D Systems Europe, Ltd., Abingdon, UK) according to the manufacturer's instructions.

Mixed Leukocyte Reaction (MLR)
Immature DC or LPS-stimulated DC were or were not treated with Silibinin (100 µM), washed with PBS, and then co-cultured with CD4 + lymphocytes purified by indirect magnetic sorting with a memory CD4 + T-cell isolation kit (Miltenyi) at 1:10 ratio. To track proliferating cells, CD4 + T-cells were intracellularly labeled with the fluorescence dye carboxyfluorescein succinimidile estere (CFSE) (Thermo Fisher Scientific, Waltham, MA, USA) which decreases after each cell division. The proliferative response was evaluated after 7 days by measuring the CFSE dilution by flow cytometry and identified by the percentage of CFSE low + cells. CFSE-unlabeled CD4 + T-cells were also analyzed by flow cytometry for their intracellular cytokine production.

Analysis of Intracellular Cytokine Production
CD4 + T-cells after stimulation with 50 ng/mL phorbol myristate acetate (PMA) and 1µg/mL ionomycin (both from Sigma) for 5 h and in the presence of Golgi Plug (BD Bioscience, San Jose, CA, USA) for the last 2 h of culture, were stained with FITC conjugated anti-human IL-17A Mab and APC/Cyanine7 conjugated anti-human IFN-γ Mab (BD Bioscience PharMingen) after fixation and permeabilization using Cytofix/Cytoperm (BD Bioscience PharMingen), according to the manufacturer's instructions. Stained cells were analyzed on a Beckman Coulter Gallios™ flow cytometer and data were analyzed with Kaluza Analysis Software v2.1 (Beckman Coulter).

Statistical Analysis
Statistical analysis was performed by the Mann-Whitney U test using GraphPad Prism (version 5.0, GraphPad Software, San Diego, CA, USA). A p-value < 0.05 was considered statistically significant.

Conclusions
Dendritic cells are specialized antigen-presenting cells that maintain immune tolerance to self-antigens by deleting or monitoring auto-reactive T-cells. Several immunosuppressive and anti-inflammatory agents interfere with phenotypic and functional DC maturation, a classical example of which is represented by corticosteroids [13]. Our data demonstrate that the flavonoid Silibinin has immunosuppressive and anti-inflammatory effects by hampering the maturation, inflammatory cytokine production, and antigen-presenting capacity of human monocyte-derived DC. Such evidence encourages the use of Silibinin as a therapeutic tool or a dietary nutritional supplement for the treatment of inflammatory/autoimmune diseases. However, further investigations in vivo are needed to support Silibinin application and therapeutic use in regard to its biological activities.
Author Contributions: M.T.P. and K.F. performed the experiments and analyzed data, M.P. and E.O. analyzed data and revised the manuscript, and D.P. provided overall supervision, designed the experiments, and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki. The project and the patients' informed consent were approved by the Review Board of the Italian National Institute of Health, Rome, Italy. The experimental protocol was performed in accordance with the guidelines and regulations provided by the local Ethical Committee (protocol no.AOOI-SS-4/01/2022 0000197).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data supporting this study's findings are available upon request.


s2fieldsofstudy: Biology Chemistry

text:
Multifunctional Polymeric Micelles for Cancer Therapy

Polymeric micelles, nanosized assemblies of amphiphilic polymers with a core–shell architecture, have been used as carriers for various therapeutic compounds. They have gained attention due to specific properties such as their capacity to solubilize poorly water-soluble drugs, biocompatibility, and the ability to accumulate in tumor via enhanced permeability and retention (EPR). Moreover, additional functionality can be provided to the micelles by a further modification. For example, micelle surface modification with targeting ligands allows a specific targeting and enhanced tumor accumulation. The introduction of stimuli-sensitive groups leads to the drug’s release in response to environment change. This review highlights the progress in the development of multifunctional polymeric micelles in the field of cancer therapy. This review will also cover some examples of multifunctional polymeric micelles that are applied for tumor imaging and theragnosis.

Introduction
Advancements in nanoscience and nanotechnology have proposed innovative ways for the treatment of diseases, especially cancer, that pose a significant challenge for clinicians [1][2][3][4]. Researchers are currently investigating a variety of nanosized drug-delivery systems for enhanced delivery of anticancer drugs, including small-molecule drugs and genes. These nanomedicines can provide effective ways to successfully overcome many drawbacks of anticancer drugs associated with poor solubility, poor pharmacokinetics (PK), non-selectivity, and toxicity. The advantages achieved by the nanomedicines studied to date are the ability to solubilize hydrophobic drugs, improved pharmacokinetics (PK), controlled drug release, biocompatibility, targetability, and responsiveness to external stimuli, enabling triggered drug release, thereby improving the overall efficacy [5][6][7][8][9][10].
Previously, specific reviews have been published on polymeric micelles for cancer therapy. For example, the review by Kaur et al., (2022) specifically focused on the clinical challenges of polymer-based micelles [11]. Similarly, Junnuthala et al., (2022) reviewed applications of micelles specifically for breast cancer therapy [12]. Similar studies have been reported earlier as well. Exemplifying such studies is the review of polymeric micelles for cancer therapy published by A Varela-Moreira et al., (2017) [13]. Here we review only the latest updates on polymeric micelles for cancer therapy.
Therefore, the present review focuses on the new developments on polymeric micelles, which have a core-shell nanostructure formed by self-assembly of amphiphilic polymers. Adding to the tumor-targeting property of polymeric micelles via the EPR effect, various modifications of polymeric micelles enabling active targeting and stimuli-sensitive release are discussed. Finally, we discuss multifunctional polymeric micelles, which are applied for tumor imaging and theragnosis. Phenyl boronic acid conjugated mPEG-b-PCL 100 nm, spherical in shape Hydrophobic, electrostatic and pendant interactions micelles as DOX carriers for enhanced drug encapsulation and controlled drug release [26] Redox-responsive diblock copolymers (mPEG-SS-PMLA) of poly(ethylene glycol) and phenyl-functionalized poly(lactic acid) with disulfide bond as the linker are synthesized to prepare PLA-based micelles 131.0 nm, spherical particles π-π interactions between Ru complex and polymer micelles PDT against 4T1 tumor model by Ru-containing polylactide MPEG-SS-PMLA@Ru micelles [27] Imidazole-bearing block polymer 200 nm, spherical particles Hydrogen bonding with DOX DOX delivery to human ovarian adenocarcinoma [28] Hyaluronic acid (HA)-conjugated curcumin (Cur) and d-α-tocopherol acid polyethylene glycol succinate (TPGS) 66 nm, spherical particles Possible hydrogen bonding, but there was no evidence justified selective drug-carrying vehicles to deliver dasatinib (DAS) to HePG2 tumors [29] mPEG-PCL~50 nm Possible hydrogen bonding, but there was no evidence provided metformin and Chrysin Delivery to Breast Cancer in Mice with improved efficacy [30] FA-PEG-PLGA~80 nm It could be hydrogen bonding interactions for the loading of two drugs MCF-7/ADR xenograft tumor inhibition by the co-delivery of DOX and SIS3 [31] PLA-PEG/SPIONS -NA Physical or hydrogen bonding could make such formulations For delivery of galactose-targeted 19-Otriphenylmethylandrographolide (RSPP050) to HepG2 cells [32] PLA-PEG NA The process of drug encapsulation is mostly operated by the hydrophobic and hydrophilic interactions.

Polymeric Micelles with Passive Targeting Function
Longer circulation allows the polymeric micelles to be accumulated in a tumor. The accumulation has been known to be preceded by an enhanced permeability and retention (EPR) effect, which is the basis of passive targeting of polymeric micelles. Growing tumors require a high amount of nutrients and oxygen, and tumors drive blood vessel growth. Tumor vasculatures grow fast, and vascular density tends to decrease as tumors grow. The resulting structural abnormality makes the tumor blood vessels highly permeable [39]. As a result, polymeric micelles can penetrate through the leakage of blood vessels and be retained in tumor tissue. This phenomenon was first reported by Matsumura and Maeda in 1986 and was named the EPR effect [40]. The cutoff size of tumor blood vessel leakage determines the diffusion of polymeric micelles in a tumor, and the cutoff size is suggested to be between 400 and 600 nm in diameter [41].
Polymeric micelles were used to deliver hydrophobic anticancer drugs to tumors using a passive targeting effect. Yu et al. designed a PEG-camptothecin conjugate (PEG-CPT) and evaluated its antitumor effect in a xenograft model of human colon cancer. The results showed that PEG-CPT provided improved biodistribution and increased the drug's stability, offering better tumor uptake with reduced side effects. PEG-CPT did not induce apoptosis in the liver and kidneys. However, PEG-CPT enhanced apoptosis induction in the tumor [42]. Notably, PEG-PLA copolymer-based micelles were approved in the Republic of Korea in 2007. Genexol ® -PM (Samyang Co., Seoul, Korea) is a polymeric micellar formulation of paclitaxel. Genexol ® -PM showed greater anticancer activity and higher tumor tissue concentration compared to paclitaxel [43]. Genexol ® -PM was approved for the treatment of breast, lung, and ovarian cancers.
Although passive targeting has been proven useful clinically, it has drawbacks. The major drawbacks of passive targeting are associated with the inaccuracy in targeting the tumor. EPR-mediated passive targeting is considerably dependent on vascular permeability. However, a tumor is induced by not only the outgrowth of the cells but also epigenetic mutation caused by physical and biological signals in a tumor's microenvironment (TME) [44]. The heterogeneity in TME, composed of various vascular conditions, contributes to the heterogeneity in EPR-mediated accumulation. The heterogeneity includes various receptor expression and interstitial fluid pressure as well as vascular permeability. For this reason, the extent of tumor accumulation varies between tumor types. According to previous studies, the vascular cutoff sizes were various between tumors, and the vasculature exhibits different porosities and permeability, even in the same tumor [45,46].
There have been new micelle systems reported recently for multifaceted applications. However, the passive EPR-assisted cancer therapy of micelles is still challenging and could be least specific to many cancers. Therefore, specific targeting functionalities should be done on such micelle structures to impart better therapeutic outcomes.

Polymeric Micelles with Active Targeting Function
As discussed in the previous section, only passive targeting depending on the EPR effect cannot fully guarantee the effective delivery of polymeric micelles to tumors. Additional approaches are required for robust tumor targeting. Cancer cells and tumor vasculature frequently exhibit enhanced expression of certain antigens or receptors compared to normal cells and tissues. Actively targeting polymeric micelles exploits this feature of cancer cells for selective accumulation of anticancer drugs in the tumor tissue. In the preparation of polymeric micelles for active targeting, the polymeric micelle surface is chemically modified by targeting ligands having a strong binding for antigens or receptors over-expressed on cancer cells. To date, various ligands have been investigated for active targeting of polymeric micelles.
Antibodies have been mostly adopted as targeting ligands for polymeric micelles, due to their diversity and specificity. Jin et al. prepared PEG-PLGA micelles conjugated with bivalent fragment HAb18 F(ab ) 2 , which binds to hepatocellular carcinoma (HCC) cells with high affinity. The cancer cells (Huh7 and HepG2) incubated with targeted micelles (DOX-loaded HAb18 F(ab )2-PEG-PLGA micelles) showed nearly 1.5 times higher cellular uptake compared to nontargeted micelles (DOX-loaded PEG-PLGA micelles). DOX-loaded targeted micelles showed higher toxicity than nontargeted micelles in a cell study using HepG2 cells. The animal study using xenograft nude mice bearing HepG2 cells shows that targeted micelles were distributed to the tumor, while the accumulation in the brain, heart, lungs, liver, spleen and kidneys was low. Furthermore, targeted micelles showed significant suppressed tumor growth compared to free DOX and DOX-loaded PEG-PKGA. The calculated inhibition rates of tumor growth (%) were 39.8%, 50.2%, and 63.9% for free DOX, DOX loaded PEG-PLGA, and targeted micelle-treated groups, respectively [47].
Peptides have been used as targeting ligands since their immunogenicity is lower compared to proteins [48]-for example, kNGR (Asn-Gly-Arg) peptide-functionalized lipidpolymer-based NPs for the targeted delivery of Paclitaxel (PTX). The micelle backbone was based on PLGA-lecithin-PEG, whereas the peptide conjugation will boost the CD13 targeting property of the NPs (Figure 1). Approximately 200-nm-sized micelles were made by a nanoprecipitation method and were found to be significantly effective in terms of higher apoptosis and cellular localization on HT-1080 (Human fibrosarcoma cell line). The in vivo results demonstrated tumor inhibition of~60% on an HT-1080 tumor model by these micelle formulations. The experimental outcome assures that such peptide-tagged micelles formulations would be highly effective against various solid malignant tissues with CD13 overexpression [49].
in the brain, heart, lungs, liver, spleen and kidneys was low. Furthermore, targeted micelles showed significant suppressed tumor growth compared to free DOX and DOXloaded PEG-PKGA. The calculated inhibition rates of tumor growth (%) were 39.8%, 50.2%, and 63.9% for free DOX, DOX loaded PEG-PLGA, and targeted micelle-treated groups, respectively [47].
Peptides have been used as targeting ligands since their immunogenicity is lower compared to proteins [48]-for example, kNGR (Asn-Gly-Arg) peptide-functionalized lipid-polymer-based NPs for the targeted delivery of Paclitaxel (PTX). The micelle backbone was based on PLGA-lecithin-PEG, whereas the peptide conjugation will boost the CD13 targeting property of the NPs (Figure 1). Approximately 200-nm-sized micelles were made by a nanoprecipitation method and were found to be significantly effective in terms of higher apoptosis and cellular localization on HT-1080 (Human fibrosarcoma cell line). The in vivo results demonstrated tumor inhibition of ~ 60% on an HT-1080 tumor model by these micelle formulations. The experimental outcome assures that such peptide-tagged micelles formulations would be highly effective against various solid malignant tissues with CD13 overexpression [49]. In general, drug delivery to brain tumors are difficult to due to the BBB (blood-brain barrier) [50][51][52][53]. To overcome such barriers, multifunctional fatty acid grafted chitosan micelles have been made for pVGF delivery to the brain and have been found to be effective by an intranasal administration. The as-made hemo-and biocompatible, 200-nmsized cationic micelles were evaluated by two routes of administration (intranasal and intravenous). The chitosan micelles were modified by the mannose, TAT peptide, and oleic acid and were found to significantly enhance VGF expression in primary astrocytes and neurons and were better when administered in an intranasal route. Both mannose and TAT ligand tags were able to enhance pVGF localization to the brain [54].
Similarly, CD44-targeted amphiphilic micelles (~154 nm) based on polyethylene glycol-block-hydroxyethyl starch-block-poly (L-lactic acid) (CD44p-conjugated PEG-b-HESb-PLA) were developed by a self-assembly technique for improving the solubility of a hydrophobic Emodin (Emo) drug for triple negative MDA-MB-231 cells. The cellular uptake and efficacy test were conducted on MDA-MB-231 cells, showing these Emo-loaded polymer micelles could be a potential nanomedicine for CD44-targeted chemotherapy [55].
The αvβ3 integrin receptors have been reported to overexpress in many tumors [56][57][58]. Targeted drug delivery to such receptors can further improve the anticancer therapy very effectively. A selective αvβ3 integrin-specific peptide ((Cyclo(Arg-Gly-Asp-D-Phe- In general, drug delivery to brain tumors are difficult to due to the BBB (blood-brain barrier) [50][51][52][53]. To overcome such barriers, multifunctional fatty acid grafted chitosan micelles have been made for pVGF delivery to the brain and have been found to be effective by an intranasal administration. The as-made hemo-and biocompatible, 200-nmsized cationic micelles were evaluated by two routes of administration (intranasal and intravenous). The chitosan micelles were modified by the mannose, TAT peptide, and oleic acid and were found to significantly enhance VGF expression in primary astrocytes and neurons and were better when administered in an intranasal route. Both mannose and TAT ligand tags were able to enhance pVGF localization to the brain [54].
Similarly, CD44-targeted amphiphilic micelles (~154 nm) based on polyethylene glycolblock-hydroxyethyl starch-block-poly (L-lactic acid) (CD44p-conjugated PEG-b-HES-b-PLA) were developed by a self-assembly technique for improving the solubility of a hydrophobic Emodin (Emo) drug for triple negative MDA-MB-231 cells. The cellular uptake and efficacy test were conducted on MDA-MB-231 cells, showing these Emo-loaded polymer micelles could be a potential nanomedicine for CD44-targeted chemotherapy [55].
The α v β 3 integrin receptors have been reported to overexpress in many tumors [56][57][58]. Targeted drug delivery to such receptors can further improve the anticancer therapy very effectively. A selective α v β 3 integrin-specific peptide ((Cyclo(Arg-Gly-Asp-D-Phe-Lys); abbreviated as c(RGDfK)) was functionalized on camptothecin (CPT). The as-made prodrug can make self-assembled NPs for precise targeting with enhanced tumor-homing capability and altogether improved anticancer efficacy as well. In vivo metabolic analysis demonstrated enhanced blood circulation for CPT by the NPs. While the control CPT can be rapidly cleared out from the body, the peptide-targeted NPs were selectively accumulated in tumor tissues, indicating that the cRGD-tagged micelles can effectively target the tumor tissues and could be useful for many other types of solid tumors as well [59].
HER2-targeted multifunctional stable micellar formulation was found to be very effective on breast cancer cells. The well-stable, pH-dependent biodegradable micelles were made by an acid-responsive cross-linking through RAFT polymerization, then a maleimide-DOX was attached to the pyridyl-disulfide-modified micelles. The developed polymers then coupled with a peptide that can bind HER2-receptors. The efficacy was revealed by experimenting on two cell lines, such as MCF-10A (HER2-negative) and SKBR-3 (HER2-positive). The specificity and selective targeting thereby enhanced apoptosis and was determined to be more on HER2-positive cells [60].
Lys); abbreviated as c(RGDfK)) was functionalized on camptothecin (CPT). The as-made prodrug can make self-assembled NPs for precise targeting with enhanced tumor-homing capability and altogether improved anticancer efficacy as well. In vivo metabolic analysis demonstrated enhanced blood circulation for CPT by the NPs. While the control CPT can be rapidly cleared out from the body, the peptide-targeted NPs were selectively accumulated in tumor tissues, indicating that the cRGD-tagged micelles can effectively target the tumor tissues and could be useful for many other types of solid tumors as well [59].
HER2-targeted multifunctional stable micellar formulation was found to be very effective on breast cancer cells. The well-stable, pH-dependent biodegradable micelles were made by an acid-responsive cross-linking through RAFT polymerization, then a maleimide-DOX was attached to the pyridyl-disulfide-modified micelles. The developed polymers then coupled with a peptide that can bind HER2-receptors. The efficacy was revealed by experimenting on two cell lines, such as MCF-10A (HER2-negative) and SKBR-3 (HER2-positive). The specificity and selective targeting thereby enhanced apoptosis and was determined to be more on HER2-positive cells [60].
Similarly, folic-acid (FA)-targeted, pH-sensitive, cell-membrane-mimicking mixed micelles were made for DOX delivery. The mixed 150-nm-sized mixed micelles, composed of P(DMAEMA-co-MaPCL) (PCD) and FA-P(MPC-co-MaPCL) (PMCF), were made by a dialysis method (Figure 2). The HeLa and MCF-7 tumor cells were significantly suppressed after being treated with these micelles, suggesting that these nanomicelles could be beneficial for FA-targeted therapy for various tumors [61]. Since phenylboronic acid (PBA) has a targeting ability towards sialic acid (SA), which is overexpressed in tumors, PBA-modified micelles would be highly effective for cancer therapy. Such an idea was established on poly(maleic anhydride) by conjugating not only PBA and F127, but also ethanolamine for doxorubicin (DOX) delivery. DOX release was acid-sensitive, and increased polymer concentration had enhanced tumor targeting, as proven on HepG2, with higher content of SA-containing glycosphingolipids than that of anti-B16. The in vivo studies on the activity of B16-bearing mice showed that the DOXloaded micelles could inhibit the tumor growth and were safer than free DOX. Thus, the PBA-modified nanopolymer micelles have potential biomedical applications due to their nanostructure and tumor-targeting ability [38].
The recent studies indicated tremendous progress on active targeted polymer micelles for cancer therapy. However, in most of the cases, detailed toxicity and safety studies are not specifically reported. Since phenylboronic acid (PBA) has a targeting ability towards sialic acid (SA), which is overexpressed in tumors, PBA-modified micelles would be highly effective for cancer therapy. Such an idea was established on poly(maleic anhydride) by conjugating not only PBA and F127, but also ethanolamine for doxorubicin (DOX) delivery. DOX release was acid-sensitive, and increased polymer concentration had enhanced tumor targeting, as proven on HepG2, with higher content of SA-containing glycosphingolipids than that of anti-B16. The in vivo studies on the activity of B16-bearing mice showed that the DOXloaded micelles could inhibit the tumor growth and were safer than free DOX. Thus, the PBA-modified nanopolymer micelles have potential biomedical applications due to their nanostructure and tumor-targeting ability [38].

Polymeric Micelles with Stimuli-Responsive Function and Their Applications in Photothermal Therapy
The recent studies indicated tremendous progress on active targeted polymer micelles for cancer therapy. However, in most of the cases, detailed toxicity and safety studies are not specifically reported.

Polymeric Micelles with Stimuli-Responsive Function and Their Applications in Photothermal Therapy
The tumor-selectivity of polymeric micelles can be strengthened by adding a stimuliresponsive property. Polymeric micelles can be engineered to respond to various stimuli and release anticancer drugs. Especially, stimuli such as low pH, hypoxia, temperature, electromagnetic waves (NIR, RF(radio frequency) etc.), and cancer-specific enzymes are commonly found in tumors. The low pH in tumors has been widely exploited to design stimuli-responsive polymeric micelles. The acidic pH in tumor results from the excess lactic acid produced by cancer cells via altered metabolism. The tumoral pH is 6.5 compared to 7.4 in the normal tissues. The decreased pH is known to promote multiple processes such as angiogenesis, metastasis, and cancer invasion [62].
Kim et al. synthesized cross-linked polymer micelles by using PEG-conjugated poly(methacrylic acid) (PEG-PMA) copolymers as templates. The cross-linking was achieved by reacting PEO-PMA block copolymers with an oppositely charged metal cation, Ca 2+ . The DOX-loaded polymeric micelles exhibited pH-sensitive release behavior with accelerated release of DOX in an acidic environment. Cytotoxic activity of DOX-loaded PEG-PMA micelles was evaluated against human ovarian cancer cells (A2780) and was compared to Doxil ® . DOX-loaded PEG-PMA micelles showed lower cytotoxicity than free DOX, which showed a higher IC50 value compared to Doxil ® . This result suggests that the sustained DOX release from the micelles results in the reduction in the cytotoxicity of DOX [63].
A thermosensitive PCL micelle was conjugated with mesoporous silica nanoparticle (MSN) by disulfide link. The DOX was loaded in both micelles and MSN, enabling a pH/redox/temperature-sensitive DOX@MSN-S 2 -F127-PCL@DOX, abbreviated as DMSFPD. When the temperature was~40 • C, F127-PCL250 (FP250), the micelle could dissociate from MSN with GSH stimuli, and the shrunken FP250 micelle enabled a sudden DOX release from both MSN and the micelle, causing cytotoxic effects on various cancer cells in vitro [64].
Compared to the single use of photodynamic therapy (PDT), it was suggested that the combination of hypoxia-sensitive therapy could improve its overall efficacy. To realize such a combined therapy, it is important to control the drug release and activate the prodrug in a safe manner. Such a system was made using an NIR-sensitive nanomicelle, ((EGylated cypate (pCy) and mPEG-polylactic acid (mPEG2k-PLA2k)) with a bioreductive prodrug (tirapazamine, TPZ). The whole system was designated as (TPZ@pCy) and made by a nanoprecipitation technique against metastatic breast cancer. Intravenously administered TPZ@pCy accumulated predominantly in the tumor, which could ablate the tumor and lung metastasis with no severe toxicity to other major organs (Figure 3) [65].  Ruthenium (Ru) complexes are known for anticancer therapy. However, the selectivity towards cancer cells is very difficult in addition to the low production of ROS for PDT. Such limitations could be overcome by micellar modification of the Ru complex. For example, a biodegradable Ru-containing polymer (poly(DCARu)) was developed for integrating two therapeutic molecules (the drug and the Ru complex) by conjugating a diblock copolymer (MPEG-b-PMCC) containing hydrophilic poly(ethylene glycol) and cyanofunctionalized polycarbonate. Upon NIR irradiation, these micelles were able to inhibit the tumor growth effectively in vivo [34]. In another work, 2-pyridone-bearing BODIPY Ruthenium (Ru) complexes are known for anticancer therapy. However, the selectivity towards cancer cells is very difficult in addition to the low production of ROS for PDT. Such limitations could be overcome by micellar modification of the Ru complex. For example, a biodegradable Ru-containing polymer (poly(DCARu)) was developed for integrating two therapeutic molecules (the drug and the Ru complex) by conjugating a diblock copolymer (MPEG-b-PMCC) containing hydrophilic poly(ethylene glycol) and cyano-functionalized polycarbonate. Upon NIR irradiation, these micelles were able to inhibit the tumor growth effectively in vivo [34]. In another work, 2-pyridone-bearing BODIPY photosensitizer was linked to polyethyleneglycol-b-poly(aspartic acid) to form a photosensitizer-1 O 2 generation, storage/release agent dual-loading system (PEG-b-PAsp-BODIPY) for cancer therapy [35]. A host-guest interaction of benzimidazole-terminated PHEMA-g-(PCL-BM) and β-CDstar-PMAA-b-PNIPAM enabled an 80 nm micelle for thermosensitive drug delivery of DOX. The lowest critical solution temperature (LCST) was between 40 and 41 • C and the DOX-loaded micelles displayed excellent anticancer activity compared to the innate DOX [36]. A 120-nm-sized, IR780-loaded, PLA-based micelle showed selective toxicity to MDA-MBA-231 cells under laser irradiation, suggesting its potential as a multifunctional theranostic agent for breast cancer treatment with increased cellular uptake, PDT, and more reliable tracking in cell-image studies [37].
PLA-based micelles were developed for Ru loading for enhanced cancer therapy. The micelles are composed of (MPEG-SS-PMLA) of poly(ethylene glycol) and phenylfunctionalized poly(lactic acid) with a disulfide bond. The MPEG-SS-PMLA had~83% Ru content, thanks to the π-π bonding among the phenyl ring and Ru complex. Under the GSH condition of 10 mmol/L, 70% of Ru was released to cause apoptosis on MCF-7 through PDT (Figure 4) [27].  It is well known that the synergy between PTT (photothermal therapy) and chemotherapy could improve overall anticancer efficacy. However, developing stable photosensitizer-loaded micelles is always challenging. A biodegradable hybrid micelles (HMs) nano system was developed for the for co-delivery of paclitaxel (PTX) and IR825. The hybrid micelle (PTX/IR825-TAT HMs) was self-assembled through hydrophobic interactions between polyethyleneimine-polycaprolactone (PEI-PCL) and TAT peptide-modified-1,2distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)5000 (TAT-PEG-DSPE). In vivo antitumor results showed that PTX/IR825-TAT HMs can selectively enter tumor tissue, enabling chemophototherapy to be effective [66].
Even though there are various reports on stimuli-responsive polymer micelles, their clinical applicability is still challenging due to the lack of long-term toxicity analyses.

Polymeric Micelles with Imaging and Theranostic Functions and Their Applications in Photodynamic Therapy
Multifunctional polymeric micelles have been extensively investigated for better tumor-specific delivery of anticancer drugs. A number of combinations of active targeting and stimuli-sensitive properties have been explored to achieve multifunctionality. Most of the polymer-drug conjugates (PDCs) have been made by click chemistry, which is catalyzed by a copper(I) reaction. In 2022, the Nobel Prize-which was shared by Carolyn R. Bertozzi In the flow cytometry study, FA-conjugated micelles showed an enhanced cellular uptake compared to nontargeting micelles. FA-conjugated micelles efficiently delivered DOX in the cells and DOX fluorescence was observed mainly in the cell cytoplasm. The cytotoxicity of DOX-loaded FA-conjugated micelles were compared with DOX-loaded micelles and free DOX. The study result showed that free DOX was more toxic than DOX-loaded micelles, since free DOX was internalized to the cell nucleus faster than DOX released from DOX-loaded micelles. However, FA-conjugated micelles exhibited a higher cytotoxicity compared to non-targeted micelles, which is consistent with its enhanced cellular uptake observed in flow cytometry [72].
Click chemistry was used for creating self-assembled polymer-drug conjugates (PDCs). Two amphiphilic PDCs were modified with PEG for loading 5-fluorouracil and coumarin, providing a unique opportunity to develop promising codelivery carriers for synergistic cancer therapy. The micelles showed anticancer efficacy on PANC-1 and BxPC-3, confirming that both coumarin and 5-fluorouracil retain their anticancer properties after conjugation with PEG [74].
Inflammatory bowel diseases ((ulcerative colitis (UC)) can be treated successfully by siRNAs by oral route. However, oral administration of naked siRNA is challenging, as it can easily be damaged by the harsh gastrointestinal tract. A nanocarrier of micelles based on MPEG-PCL-CH2R4H2C was used for oral delivery of siRNA to treat a dextran sodium sulfate-induced UC model [75]. The preliminary results are promising and could be extended for colorectal cancers as well. For example, capecitabine (CAP) was loaded with pH-responsive beta-cyclodextrin (CD)-based micelles for colon cancer therapy [76]. Similarly, there have been wide varieties of multifunctional polymer micelles for magnetic hyperthermia application as well [77]. Such a study by Cheng et al., (2022) reported multifunctional micellar theranostic systems for cancer therapy. The hybrids consist of glucose and TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl) at the distal ends of PEO-b-PLLA block copolymer (glucose-PEO-b-PLLA-TEMPO), which can encapsulate CUDC101 and IR780 effectively. The specific core-shell rod structure formed by the designed copolymer gives TEMPO radicals excellent stability against reduction-induced magnetic resonance imaging (MRI) silence. The glucose enables tumor targeting, whereas the TEMPO can have trimodal imaging via MRI, photoacoustic imaging, and fluorescence. Efficient delivery of CUDC101 and IR780 is achieved to synergize the antitumor immune activation through IR780-mediated photodynamic therapy (PDT) and CUDC101-triggered CD47 inhibition, showing M1 phenotype polarization of tumor-associated macrophages (TAMs). More intriguingly, this study demonstrates that PDT-stimulated p53 can also re-educate TAMs, providing a combined strategy of using dual tumor microenvironment remodeling to achieve the synergistic effect in the transition from a cold immunosuppressive to a hot immunoresponsive tumor microenvironment [78].
Recently, there have been huge improvements in theranostic approaches using polymerbased micelles; however, there must still be careful analysis of their long-term applicability to improve cancer therapy.

Clinical Trials
Even though there are many reported studies related to polymer micelles in the clinical trials, some of the recent ones are listed in Table 2. It was found that most of such studies are focused on either docetaxel and paclitaxel drug formulations as passively targeted formulations. As per Table 2, the only formulation with active targeting under clinical trial is the one with cetuximab/5-FU (5-flouro uracil) combined with the previous clinical trial of NC-6004.
The NC-6004 clinical-drug-based polymer micelle nanotechnique has been studied. This specific drug has a particle size of~30 nm and the basic reaction chemistry is based on a polymer-metal complex between polyethylene glycol poly (glutamic acid) block copolymers (PEG-P(Glu)) [79]. This ultrafine-sized clinical drug can be tumor-homed through the EPR path [40]; additionally, it was reported that NC-6004 could be in blood circulation longer than its original intact form (conventional cisplatin), which has a short half-life, thanks to the PEG shell on the NC-6004, which can sustain blood circulation significantly higher than the conventional drug [78]. Such beneficial properties of NC could give rise to enhanced pharmacokinetic profile with increased AUC while decreasing the C max , availing a safer therapeutic window than the intact cisplatin. Laboratory experiments confirmed the safety of NC-6004 in terms of lowering the nephrotoxicity and neurotoxicity more than the intact cisplatin at an equivalent dose [80]. Several other reported clinical trials using polymer micelle particles are shown in Table 2.

Conclusions
In this review, we have discussed various examples that cover a wide range of polymeric micelles applied in the field of anticancer drug delivery. Passive targeting with polymeric micelles has great potential; however, TME (tumor microenvironment) heterogeneity limits the potential. To overcome this, various functionalities were employed in polymeric micelles. The block copolymers forming micelles can be chemically modified, facilitating the employment of various functionalities. Due to this, polymeric micelles could be further developed for active targeting and triggered drug release in response to special cues provided by the tumor microenvironment. However, the design of functional polymeric micelles should consider various factors, including targeting, drug release, therapeutic efficacy, biodistribution, and systemic toxicity. In spite of rapid benchside developments, the translation of polymeric micelle to the bedside has been less impressive. Only a few micelle-based drug delivery systems have been approved by the Food and Drug Administration (FDA). The slow bench-to-bedside translation is because of several challenges that need careful research. The main reason is the lack of preclinical tissue culture systems that mimic biological conditions to predict the therapeutic activity and targeting efficiency performance of polymeric micelles. Thus, new evaluation tools should be further developed to accelerate the clinical translation of polymeric micelles. With the development of multifunctional polymeric micelles, the structure of the polymeric micelles becomes more complex, leading to difficulties in reproducible synthesis and scale-up. Impediments to clinical translation can originate from the challenge in developing a robust manufacturing process meeting the regulatory requirements.
In spite of the hurdles mentioned above, more well-designed multifunctional micelles will be investigated, and there will be more accumulated cases translated to clinical trials. In addition, a micellar drug-free therapeutic area should also be well utilized with new functionalities in the coming days. The multidisciplinary field related to chemistry, medicinal science and clinical research holds the promise of delivering breakthroughs in polymeric micelle-based drug delivery, facilitating their move from concept to clinical fields. However, there is long way to go for such establishments because of the lack of detailed toxicity assessments on novel polymer micelles. Therefore, newly developed micelles should be thoroughly checked for toxicity and long-term human applications.


The complete chloroplast genome sequence of begonia (Begoniaceae)

Abstract Begonia L. (Begoniaceae) is the sixth largest genus in the world which consists of more than 2,039 species. Many species of Begonia have highly ornamental leaves and flowers, so they are mainly used for ornamental purposes, and some species can also be used as medicines or vegetables. Begonia gulongshanensis is a newly discovered species in 2018 which occurs exclusively in Jingxi county in Southern China, however, there are few studies on the molecular biology and phylogeny of this species currently. Therefore, we report its complete chloroplast genome sequence for the first time, hoping to provide a foundation for its future phylogenetic analysis. The chloroplast genome of B. gulongshanensis was 169,153 bp in size, which contained a large single-copy region of 75,998 bp, a small single-copy region of 18,063 bp, and two inverted repeat regions of the same 37,546 bp. The total GC content was 35.51%. The genome encodes 42 transfer RNA genes, 8 ribosomal RNA genes and 90 protein-coding genes. The phylogenetic analysis indicated that the genetic relationship between B. gulongshanensis and the other three begonias was very close, but there was still certain distance.

Begonia L. (Begoniaceae) was mainly distributed in tropical and subtropical areas (Tian et al. 2018), and 2,039 species in Begonia L. have been published (Hughes et al. 2015-Prensent). As the sixth largest genus in the world, it contains tuberous, semi-tuberous, moniliform, rhizomatous, trailing (scandent), and shrub-like species, and there is a huge diversity in habitat, habit, plant size, leaf type, flower and fruit morphology between different species (Tian et al. 2018). Many species of Begonia have highly ornamental leaves and flowers, so they are mainly used for ornamental purposes, and some species can also be used as medicine or vegetable (Guan et al. 2007). In the last 20 years, many new species have been discovered because of increasing attention from people (Tian et al. 2018;Dong and Liu 2019;Van et al. 2021) predicted that the number of species of Begonia may actually in total between 2000 and 2500. However, the classification of species in Begonia is still controversial, due to the traditional classification method is mainly based on morphological observations. In recent years, taxonomists have solved some taxonomic problems through a combination of morphology and molecular technology, but there are still a large number of species that lack molecular data, and there is still a lot of work remains to be done.
Begonia gulongshanensis is a newly reported species in 2018 which occurs exclusively in Jingxi county of Guangxi province in Southern China (Chen et al. 2018). It is only grows on the moist surface of shady cliffs at the entrance to shallow caves in a deep valley, with a white area on the leaves (Chen et al. 2018). Due to the rare distribution in the wild, B. gulongshanensis was listed as class II protected plant in the List of National Key Protected Wild Plants in September 2021 (National Forestry and Grassland Administration & Ministry of Agriculture and Rural Affairs of the People's Republic of China 2021). So far, there have been few studies on the molecular and phylogeny of this species. The purpose of this study was to complete the sequencing of the chloroplast genome of B. gulongshanensis and study its phylogeny, so as to provide reference for the future research on the genetic relationship between it and other Begonia species, and provide a foundation for its conservation, exploitation and utilization, and to provide reference for subsequent intraspecific classification of Begonia.
We collected the leaf samples of B. gulongshanensis from Jingxi county of Guangxi province (106 39'30 0 'E, 23 0'35 0 'N, 550 m above sea level). The collected reference samples were deposited in the Flower Research Institute, Guangxi Academy of Agricultural Sciences (http://www.gxaas.net/), and the voucher number was GXAAS-B00158 (collected by Jinye Zhou, ahzhoujy@163.com). The Illumina NovaSeq 6000 platform was used to paired-end sequencing of extracted genomic DNA, and the sequencing read length was 150 bp. Then, we assembled the chloroplast genome by SPAdes v3.10.1 (Bankevich et al. 2012), finally, annotated the chloroplast genome B. gulongshanensis by CPGAVAS 2 (Shi et al. 2019). We submitted the assembled chloroplast genome and the annotations to GenBank, and obtained the accession number MZ671995. In order to construct the phylogenetic tree of B. gulongshanensis, another 21 species (3 species from Begonia L., and 18 species from other families of Rosanae) were compared and analysised by via MAFFT v.7.271 (Katoh et al. 2005) and RAxML v8.0 (Stamatakis 2014).
The assembled chloroplast genome of B. gulongshanensis was 169,153 bp in size, with a typical quarter structure which contained a large single-copy region (LSC) of 75,998 bp, a small single-copy region (SSC) of 18,063 bp, and two inverted repeat regions (IRA and IRB) of the same 37,546 bp. The total GC content is 35.51%. The annotated results showed that there were 140 genes in B. gulongshanensis chloroplast genome, including 42 transfer RNA genes (tRNAs), 8 ribosomal RNA genes (rRNAs) and 90 protein-coding genes (PCGs). The phylogenetic analysis showed that Begoniaceae was closest to Cucurbitaceae and Corynocarpaceae, which also belong to Cucurbitales, followed by Rosales, and had the most distant with Fabales. Although B. gulongshanensis was clustered with the other three begonias, there was still a certain distance between them. We speculated that this was because the B. gulongshanensis belonged to the section Coelocentrum while the other three begonias belonged to the section Platycentrum (Figure 1).

Data availability statement
The complete sequence of the chloroplast genome of B. gulongshanensis have been deposited in GenBank at https://www.ncbi.nlm.nih.gov/, and the accession no. was MZ671995. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA770846, SRR16311536, and SAMN22241481, respectively.